ES2345734T3 - PROCESS FOR THE PREPARATION OF OPTICALLY ACTIVE CHIRAL AMINES. - Google Patents

Abstract

The present invention relates to the production of optically pure secondary amines, which can be used as intermediate products in a synthesis of for instance pharmaceutical products.

Description

Process for the preparation of chiral amines optically active

The present invention is related to a process for the preparation of optically chiral amines
active.

Chiral amines have an important role in the pharmaceutical, agrochemical and chemical industry. Frequently they are used as intermediaries or synthons for the preparation of various physiologically active substances, for example pharmaceutically active, such as cephalosporin derivatives or pyrrolidine In a large number of different applications of chiral amines, just a concrete optically active form, the enantiomer (R) or (S) possesses the desired physiological activity. Therefore, there is a clear need to provide processes for the preparation of chiral amines in optically form active

These needs are partially met by preparing chiral amines by crystallization of diastereomeric salts by the addition of chiral carboxylic acids (Breuer et al ., Angewandte Chemie (2004) 116, 806-843). Other chemical methods use enantioselective synthesis through the reduction of proquiral precursors with double C = N bonds.

In addition, stereoselective cleavage of racemates using several enzymes, such as proteases, amidases or lipases (Bornscheuer and Kazlauskas, Hydrolases in Organic Synthesis (2005), Wiley-VCH Weinheim) is known. It is also known that specific transaminases, ie α-transaminases, which include α-amino acid aminotransferases, are suitable for the preparation of optically pure amino acids (Bartsch et al ., Appl. Environm. Microbiol. (1996) 62, 3794-3799, Cho et al ., Biotechnol. Bioeng. (2003) 83, 226-234, JP 011 53084 A2 (1998), JP 633 04986 A2 (1988), PE 0 248 357 A2 and Ziehr et al . , Biotechnol. Bioeng. (1987) 29, 482-487).

Shin and Kim (Biotechnology and Bioengineering, 1999, 65 (2), 206-211) describe a process for the asymmetric synthesis of chiral amines with an ome-transaminase ( Vibrio fluvialis JS17).

Hwang and Kim (Enzyme and Microbial Technology, 2004, 34, 429-436) describe a process for produce optically active chiral amines by reaction of an amino acceptor and an amino donor in the presence of a ome-transaminase.

Cho et al . (Biotechnology and Bioengineering, 2003, 81 (7), 783-789) describe a simultaneous synthesis of an enantiomerically pure (S) -amino acid and (R) -amines using coupled transaminase reactions.

However, these previous processes have Several disadvantages. Although enzymatic processes usually they use favorable moderate conditions, unlike the classical methods, and get a reasonable stereoselectivity, these regularly use enzymes, whose specificity of substrate, enantioselectivity and / or conversion rates are not high enough to apply to industrial processes. In addition, one of the most important disadvantages of using transaminases for the preparation of optically active amines the represents the frequently observed phenomenon of inhibition of substrate and product. Therefore it is one of the objectives of the present invention provide an improved process for preparing optically active chiral amines, in particular a process with an improved substrate specificity, an enantioselectivity improved and in particular that allows a conversion of educts of up to 100%.

The present invention solves the problems technicians underlying the present invention by providing a process for the preparation of an optically active chiral amine and pyruvate as an α-ketone by-product that comprises a) providing an amino and alanine acceptor as a donor amino, b) react the amino acceptor and the amino donor with a selective transaminase of (R) or (S), c) obtain the chiral amine desired optically active and pyruvate as a byproduct α-ketone and d) remove pyruvate from the reaction mixture with a decarboxylase. According to a preferable embodiment of the present invention, in one step Optional additional subsequent process, the chiral amine optically active obtained in steps c) and d) it is isolated and purify from the reaction mixture obtained in steps c) and d).

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The reaction of the present invention follows the principle of the following scheme:

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one

Therefore, the present invention provides a process for asymmetric synthesis of chiral amines by the use of at least one transaminase for transamination of an amino group from alanine as an amino donor to a amino acceptor, thus forming the desired product. Depending on the preference of the specific transaminase enantiomer used, an optically active chiral amine is obtained from the configuration desired optic, that is the enantiomer (R) or (S). Thus, use in an embodiment of the present invention a Selective transaminase of (S) for asymmetric synthesis generates the desired enantiomer (S) of the chiral amine while using in another embodiment of the present invention a transaminase Selective of (R) generates the desired (R) enantiomer. In addition to the optically desired active amines, the reaction results in pyruvate as an α-ketone by-product at from the amino donor used and possibly from the amino acceptor and unconverted amino donor.

In the context of the present invention, a Transaminase is a pyridoxalphosphate dependent enzyme that catalyzes the transfer of amino groups. The transaminases are classified as E.C. 2.6.1.X. In one embodiment particularly Preferable of the present invention, the transaminase is a selective transaminase of (R) or (S), in particular in a preferable embodiment is a ome-transaminase.

In the context of the present invention an [omega] transaminase is an enzyme preferably with the classification code EC2.6.1.18. These aminotransaminases are characterized in that they mainly use amines as substrates. These enzymes are also characterized by showing an equilibrium constant of reactions catalyzed by?-Transaminases that is greater than 1.?? Transaminases that can be used in accordance with the present invention are described for example in Iwasaki et al ., Biotechnol Lett. (2003) 25, 1843-1846, Shin et al ., Biotechnol. Bioeng (1997) 55, 348-358, Shin and Kim, Book of Abstracts, 217th ACS National Meeting, Anaheim, Calif., March 21-25, (1999) 180, Shin and Kim, Biosc. Biotechnol Biochem (2001) 65, 1782-1788 and Shin and Kim, Biotechnol. Bioeng (1998) 60, 534-540.

Therefore, in a preferable embodiment of the present invention, the selective transaminase of (R) or (S), in particular the selective β-transaminase of (R) or (S) used in the present process is a selective transaminase of (R) or (S), in particular a selective γ-transaminase of (R) or (S) obtained from Vibrio fluvialis , in particular from strain JS17. In a more preferable embodiment, the selective transaminase of (R) or (S) is from Alcaligenes denitrificans , in particular of strain Y2k-2. In a more preferable embodiment, the selective transaminase of (R) or (S) is from Klebsiella pneumoniae , in particular from strain YS2F. In a more preferable embodiment, the selective transaminase of (R) or (S) is from Bacillus thuringiensis, in particular strain JS64. For the nomenclature of the strains see Shin and Kim, 1998, mentioned above. Of course, the present invention also includes under the term selective transaminase of (R) or (S), and in particular selective β-transaminase of (R) or (S), an extract of an organism, such as a microorganism or a cell, which contains a selective transaminase of (R) or (S), in particular a selective? R-transaminase of (R) or (S), or a living or dead cell or microorganism comprising itself a selective transaminase of (R) or (S), in particular a selective? R-transaminase of (R) or (S). Such microorganism or cell or extract or enzyme transaminase selective of (R) or (S) can be used in immobilized or non-immobilized form. The selective transaminase of (R) or (S), in particular the selective β-transaminase of (R) or (S), can also be a selective transaminase of (R) or (S) obtained recombinantly, or of natural or genetically modified occurrence, in particular a selective? R-transaminase (R) or (S), which is partially or completely encoded by a nucleic acid sequence or a derivative thereof contained in one of the organisms previously identified or equivalent to these.

In the context of the present invention the term optically active chiral amine is related to the same subject or matter as the term chiral amine enantiomerically active. These particular terms refer to to a preparation that is essentially pure, and in one embodiment even more preferable free of the unwanted enantiomer. In accordance with this, an optically active chiral amine essentially comprises a  excess of an enantiomer or even consists only of a enantiomer

In particular, in the context of this invention, an optically active chiral amine possesses purity optics of at least 70%, in particular more than 90% and at best of cases> 99%.

In the present invention, optical purity is provides in% excess of one enantiomer with respect to the other enantiomer Therefore, the optical purity in% is the ratio of the difference between the concentrations of enantiomer (R) and of (S), and the sum of the concentrations of both enantiomers (optical purity of A in% = ([A] - [B]): ([A] + [B]) x 100, in which A and B represent the concentrations of the enantiomers (R) and (S) or vice versa).

In the present invention, it is preferable that the amino acceptor becomes the desired chiral amine in a conversion of at least 40, 50, 60, 70, 80, 90, 95 and in particular 100% The concentrations to analyze the optical purity and the conversion can be determined for example by chromatography of gases (GC) or photo- or fluorometric methods.

In the context of the present invention a amino acceptor is a molecule capable of accepting an amino group transferred from an amino donor by a selective transaminase of (R) or (S), in particular a selective? R-transaminase of (R) or (S). In a particularly preferable embodiment of the present invention, The amino acceptor contains a ketone functionality. In a particularly preferable embodiment of the present invention the amino acceptor is selected from the group consisting of acid phenylpyruvic, a salt thereof, pyruvic acid, a salt of same, acetophenone, 2-ketoglutarate, 3-oxobutyrate, 2-butanone, 3-oxopyrrolidine (3-OP), 3-pyridylmethyl ketone (3-PMK), ester 3-oxobutyric acid ethyl (3-OBEE), methyl acid ester 3-oxopentanoic (3-OPME), N-1-boc-oxopiperidinone, N-1-boc-3-oxopyrrolidine (B3OP), 3-oxo-piperidine, alkyl-3-oxo-butonoates,  Methoxyacetone and 1-Oxotetralone.

In a particularly preferable embodiment the amino acceptor is B3OP.

In the context of the present invention a amino donor is a molecule capable of providing an amino group to an amino acceptor using a selective transaminase of (R) or (S), in particular an [omega-transaminase] selective of (R) or (S).

They are amino donors the β-alanine, alanine, in particular D, L-Alanine, L-Alanine or D-Alanine Ketone products are acid phenylpyruvic, a salt thereof, pyruvic acid, a salt thereof, glyoxylic acid, a salt thereof, acetophenone, 2-ketoglutarate, acetone, 3-oxobuti-while, 2-butanone, 3-oxopyrrolidine (3-OP), 3-pyridylmethyl ketone (3-PMK), ethyl acid ester 3-oxobutyric (3-OBEE), ester 3-oxopentanoic acid methyl (3-OPME), N-1-boc-3-oxopiperidinone Y N-1-boc-3-oxopyrrolidine (B3OP) or a salt, for example a chloride, of any of the same. The by-product α-ketone according With the present invention it is pyruvate.

In accordance with the present invention the donor amino is alanine, in particular L-alanine.

In another preferred embodiment, the present invention is related to a process for the preparation of an optically active chiral amine that is selected from the group of amines with an optically active amino group, in particular amines with alkyl groups, branched alkyl groups or arylalkyl groups. In particular, these amines, in particular the mono- or bicyclic amines, are in particular cyclic amines of 5 to 6 members, or heterocyclic hydrocarbons S-, O- or N-substituted, or aromatic amines, in particular aromatic amines substituted with alkyl or alkoxy. In a preferable embodiment, the chiral amines obtained are selected  from the group consisting of phenylalanine, alanine, 3-aminopiperidine, alkyl-3-amino-butanoates, 3-aminopyrrolidine (3-AP), 3-pyridyl-1-ethylamine (3-PEA), N-1-boc-3-aminopyrrolidine (B3AP), 3-aminobutyric acid ethyl ester (3-ABEE), methyl acid ester 3-aminopentanoic acid (3-APME), α-methyl-benzylamine (α-MBA), 1-aminotetralin, α-methyl-4- (3-pyridyl) -butamine, glutamate, β-aminobutyrate, sec-butylamine, methoxyisopropylamine, derivatives of 3-aminopyrrolidine, 1-N-Boc-3-aminopiperidine, cephalosporin and cephalosporin derivatives.

In a particularly preferable embodiment, The present invention, therefore, envisages reacting 3OP with a selective transaminase of (S) or (R) and alanine as an amino donor to obtain 3AP (S) or (R) optically active.

In a more preferable embodiment, the present invention provides for reacting 3-PMK with a selective transaminase of (R) or (S) and alanine as amino donor to obtain 3-PEA (R) or (S) optically active.

In another preferred embodiment of the present invention, the invention provides for reacting 3-OBEE with a selective transaminase of (R) or (S) and alanine as an amino donor to obtain 3-ABEE (R) or (S) optically active.

In another preferred embodiment, the invention plans to react 3-OPME with a transaminase selective of (R) or (S) and alanine as an amino donor to obtain 3-APME (R) or (S) optically active.

In another preferred embodiment, the invention envisages reacting B3OP with a selective transaminase of (R) or (S) and alanine as an amino donor to obtain B3AP (R) or (S) optically active

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In a particularly preferable embodiment the invention is related to a reaction between B3OP and the donor amino alanine, in the presence of a transaminase to obtain B3AP optically active and pyruvate, in which the reaction is carried out at a pH of 5.0 to 9.5, preferably 6.0 to 7.0, in particular of 6.0 to 6.9, for a period of 30 to 70 minutes, in particular 40 to 65 minutes, in particular 50 to 60 minutes

In a particularly preferable embodiment, said transaminase is a selective transaminase of (R). In other preferable embodiment, said transaminase is a transaminase selective of (S).

In a preferable embodiment, said reaction of B3OP with the donor amino alanine is performed in the presence of al minus a pyruvate decarboxylase (PDC). In another embodiment preferably, said reaction of B3OP with the amino donor amino alanine in presence of at least one pyruvate decarboxylase is performed while simultaneously gaseous nitrogen is introduced into the reaction mixture for the removal of acetaldehyde obtained from  pyruvate formed by action of the PDC.

In another preferred embodiment, said reaction of B3OP with the amino donor amino alanine in the presence of at least one pyruvate decarboxylase is performed in the presence of at least one alcohol dehydrogenase (ADH) for the elimination of acetaldehyde obtained from pyruvate formed by action of the PDC.

In another preferred embodiment, said reaction of B3OP with the donor amino alanine, in the presence of at least one pyruvate decarboxylase is performed while entering simultaneously nitrogen gas in the reaction mixture, in the that at least one alcohol dehydrogenase is present in the medium of reaction to remove acetaldehyde obtained from pyruvate formed by action of the PDC.

In another preferred embodiment of the present invention, the invention provides for reacting acetophenone with a selective transaminase of (R) or (S) and alanine as amino donor to obtain α-MBA (R) or (S) optically active

In another preferred embodiment, the present The invention provides for reacting as an amino acceptor, in particular mono- or bicyclic, cyclic hydrocarbons of 5 to 6 members that contain oxo or heterocyclic groups S-, O- or N-substituted or aromatic, in particular aromatic substituted with alkyl or alkoxy, with alanine as amino donor and a selective transaminase of (R) or (S) to obtain amines optically active, in particular mono- or bicyclic amines, in particular cyclic amines of 5 to 6 members or hydrocarbons S-, O- or N-substituted heterocyclics or amines aromatic, in particular alkyl substituted aromatic amines or alkoxy, in particular in (S) or (R) form.

In a particularly preferable embodiment of the present invention, the amino acceptor and the amino alanine donor are reacted with the selective transaminase of (R) or (S) in aqueous medium, for example a physiological buffer. In one embodiment particularly preferable the transamination reaction is performed at a pH in the range of 5.0 to 9.5, from 5 to 9, in particular from 7 to 8.5. The invention provides, in one embodiment particularly preferably, react the amino acceptor and the amino donor to a pH value between 6.0 and 7.0, preferably between 6.0 and 6.9.

In a particularly preferable embodiment, the reaction is carried out in a temperature range of 10 to 65 ° C, preferably from 20 to 50 ° C, in particular from 18 to 25 ° C, preferably from room temperature or from 34 ° C to 39 ° C and in particular at 37 ° C. In another preferred embodiment of the present invention, the amino acceptor and the amino donor are provided in a molar ratio of 1:50 to 1: 200, in particular 1:50 to 1: 100, in particular from 1: 100, and in particular from 1: 1 to 1: 5, in particular from 1: 1 to 1: 2. In a preferable embodiment of the present invention The enzymatic activity can be from 1 to 20,000 µmol / min.

In another preferred embodiment, the reaction is performed for a reaction time of 30 to 70, preferably 40 to 65, in particular 50 to 60 minutes.

Therefore, in accordance with this invention is provided a process for the preparation of an amine  optically active chiral according to the above mentioned, which means accordingly that in a first process step a) an amino acceptor and as a donor are provided amino alanine, in a second step of the process b) are made react the amino acceptor and the amino donor with at least one selective transaminase of (R) or (S), preferably a ? transaminase, in a third step of the process c) an optically pure chiral amine and pyruvate are obtained as α-ketone by-product, and in which in one step additional process d) the ketone by-product, in particular the α-ketone by-product, obtained in step c)  it is removed from the reaction mixture obtained by a reaction with an enzyme, which means by enzymatic cleavage using a decarboxylase.

In a particularly preferable embodiment, the ketone product, that is, pyruvate, obtained in step c) is removed by reaction with a pyruvate decarboxylase (PDC), for example Saccharomyces cerevisiae, Zymomonas mobilis or Zymobacter palmae , preferably thus producing acetaldehyde and CO2.

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by the action of a PDC and in which the acetaldehyde thus formed is removed for example through a chemical, enzymatic or physical treatment.

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by action of a PDC and in the that the acetaldehyde thus formed is removed for example by nitrogen gas feed into the reaction mixture, preferably by feeding said gaseous nitrogen continuously in the reaction mixture, to remove the acetaldehyde of the reaction mixture.

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by action of a PDC and in the that the acetaldehyde thus formed is removed by reacting the acetaldehyde with at least one alcohol dehydroxygenase (ADH) to remove the acetaldehyde from the reaction mixture and convert it into ethanol.

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by action of a PDC and in the that the acetaldehyde thus formed is removed by applying a pressure reduced to the reaction mixture.

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by action of a PDC and in the that the acetaldehyde thus formed is removed by reactions chemical

In another preferred embodiment, the invention It is related to a process, in which the ketone product is say pyruvate, obtained is eliminated by action of a PDC and in the that the acetaldehyde thus formed is eliminated by feeding nitrogen gas in the reaction mixture, preferably feeding said gaseous nitrogen continuously in the reaction mixture, and in which additionally reacts acetaldehyde with al minus one alcohol dehydroxygenase (ADH) to eliminate the Acetaldehyde of the reaction mixture and convert it to ethanol.

In another preferred embodiment of the present invention, the ketone product, that is pyruvate, obtained in the step c) is continuously removed from the reaction mixture.

These particularly preferable embodiments provide the advantage of obtaining a conversion rate particularly high, since the ketone product as a byproduct, that is pyruvate, from the present process it is eliminated from the equilibrium reaction The reaction is forced in the direction of the products, thus providing high stereoselectivity and a Very high conversion to the desired products.

The present invention is also related with processes for the preparation of compounds physiologically assets or their precursors and / or intermediaries in the production of themselves, in particular selected from the group of derivatives of 3-aminopyrrolidine, cephalosporin, derivatives of cephalosporin, heterocyclic boronic acid, L-dihydroxyphenylalanine (LDopa), α-methyldopa, D-phenylglycine, β-hydroxyphenylglycine, phosphinothricin, pyrimido derivatives and pyrrolidone derivatives, in which it is used any of the processes of the present invention above identified. In the context of the present invention, a compound  physiologically active is a compound that is physiologically active in plants, animals, humans, yeasts or microorganisms, such as protozoa, bacteria or viruses, meaning that it interacts with the body's metabolism

Other preferable embodiments of the present invention are those described in the subclaims.

The present invention is illustrated in more detail. in the following examples and in the accompanying figures.

The accompanying figures illustrate the present invention

Figure 1 shows a layer chromatogram fine.

Figure 2 shows the relative activity of the flu-TA of V. fluvialis as a function of the pH value.

Figure 3 shows the relative reduction of the B3AP concentration based on incubations with several substances

Figure 4 shows the relative reduction of the B3AP conversion in the presence of pyruvate and acetaldehyde.

Figure 5 shows the conversion of B3OP to B3AP over time at various pressures.

Figure 6 shows the relative conversion of B3OP to B3AP in the presence of several PDCs.

Figure 7 shows the effect of an increase in Alanine concentration and an increase in PDC concentration in the asymmetric synthesis of B3AP.

Figure 8 shows the conversion of B3OP to B3AP at increased alanine concentrations.

Figure 9 shows the conversion dependent on Relative PLP from B3OP to B3AP.

Figure 10 shows the relative conversion of B3OP to B3AP dependent on the presence of N2.

Figure 11 shows the relative conversion of B3OP to B3AP in the presence of an ADH.

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Example 1 Asymmetric synthesis of B3AP

Asymmetric synthesis of B3AP was performed in 1.5 ml reaction tubes. B3OP was used as amino acceptor in a concentration of 5 mM (7.5 µmol). The concentration of Amino donor used L-alanine was 5 mM. The reagents and reaction conditions used are evident at from Table 1 below.

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TABLE 1 Reaction conditions for asymmetric synthesis of (S) -B3AP using a (S) -? Transaminase to transaminate the group amino from alanine to B3OP

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2

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The buffer used was 50 mM sodium phosphate, pH 7. TA7 indicates the γ-transaminase of Vibrio fluvialis (Jülich Fine Chemicals, Germany). TA8 indicates the W transaminase of Alcaligenes denitrificans (Jülich Fine Chemicals, Germany). As lactate dehydrogenase an extract of Escherichia coli was used . In addition, NADH was used at a final concentration of 10 mM. The concentration of pyruvate decarboxylase was varied. 1.5 units (20 µl) and 15 units (200 µl) of Saccharomyces cerevisiae pyruvate decarboxylase (PDC1) were used. 2 units were used
(3.4 µl) and 20 units (34 µl) of Zymomonas mobilis pyruvate decarboxylase (PDC2).

TABLE 2 Conversion and optical purity obtained using TA8 for asymmetric synthesis of B3AP. The calculations were based on a GC analysis (+/- 5%)

3

Referring to Table 2 above, it is clear that in each of the six series using the TAB? Transaminase, a very high degree of optical purity of the (S) -B3AP obtained could be achieved. It was also observed that regardless of whether TA7 or TA8 was used, the degree of conversion was only moderate, if the reaction equilibrium was not influenced (series 1). Using alanine in an excess of 10-50 times only the conversion is slightly improved. In series 3, 4, 5 and 6 the ketone product of the reaction, that is pyruvate, was removed during the transamination reaction of the equilibrium reaction. The use of TA8 together with E. coli lactate dehydrogenase (series 6) resulted in an extremely improved degree of conversion while maintaining and even improving enantioselectivity. Essentially the same is true for the enantioselectivity provided by the pyruvate decarboxylase of Zymomonas mobilis (series of 3 to 5). PDC1, however, only slightly increases the conversion, PDC2 increases the conversion rate moderately (series 4) if it is reacted for 24 hours while in a 72 hour reaction (series 5) the conversion is drastically improved. All reactions took place for 24 hours except series 5, which lasted 72 hours.

The Figure shows the thin layer chromatogram of the reactions carried out according to Table 1. "A" indicates the? -Transaminase of Alcaligenis denitrificans and? V? The? -Transaminase of Vibrio fluvialis . "K" indicates the series 1 that uses only TA7 or TA8 (series 1). PDC1 indicates the series with Saccharomyses cerevisiae pyruvate decarboxylase (series 3), LDH the Escherichia coli lactate dehydrogenase series (series 6) and PDC2 the Zymomonas mobilis pyruvate decarboxylase series (after 24 and 72 hours) (series 4 and 5). Therefore, the results clearly show that the production of (S) -B3AP from the B3OP prochemical ketone can be carried out with a very high enantioselectivity. Using the? -Transmines as the only enzymes in the preparation process, however, a moderate conversion is achieved. This moderate conversion rate can be greatly improved if pyruvate is removed from equilibrium, in particular using lactate dehydrogenase or pyruvate decarboxylase. Using pyruvate decarboxylase has the advantage, among others, that cofactor recycling (NADH) is not necessary. It also advantageously provides the enzymatic elimination of pyruvate with PDC and therefore provides the additional advantage of eliminating or preventing product inhibition (ketone product) and pulls the reaction equilibrium to the right, achieving greater conversion (ideal case of 100%)

Series 6 does not belong here illustration.

Example 2 Activity pH dependence β-transaminase in the conversion reaction from (S) - αMBA to acetophenone (does not belong to this illustration)

The synthesis was carried out in a quartz cuvette using 50 µl of 100 mM pyruvate, 4 units / ml of Vibrio fluvialis [ TA] (from here also Vfl) (12 µl) and 388 µl of phosphate buffer 50 mM sodium with pH variations from pH 6.0 to pH 7.4 in 0.2 unit steps. The reaction was started with 50 µL of 100 mM (S) -αMBA as an amino donor and the increase in absorption was measured at between 250 and 260 nm. The increase in absorption is due to the acetophenone formed. The other substrates do not contribute significantly to absorption, so that the speed of the reaction can be determined by the measurement of acetophenone absorption. The value reached at pH 7.4 was taken as 100% and the relative activity in the rest of the pH values was calculated as evident from Figure 2. Figure 2 shows the relative activity of the? -AT of V. fluvialis in relation to a given pH value.

Figure 2 shows that at lower pH values, as 6.0, 6.2 or 6.4 there is still considerable activity, for example 11% at pH 6.0. Therefore, this result demonstrates that even at a low pH, it is possible to obtain an activity significant transaminase, allowing to react the substrates at a lower pH, which in turn allows to increase the conversion using a PDC, which is sensitive to pH values superior.

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Example 3 Asymmetric synthesis of B3AP at different pH values

In this example, the synthesis is shown asymmetric B3AP from alanine and B3OP in the presence and absence of a pyruvate decarboxylase (PDC).

For each pH value, 6.0, 6.4 and 7.0, three series of experiments were performed. Series 1 used the PDC of Zymomonas mobilis (wild cell extract), Series 2 used that of Zymobacter palmae (recombinant in E. coli ) and Series 3 was a control without PDC, which uses only transaminase. To obtain comparable results, the activities of both PDCs have been determined at pH 6 with an alcohol dehydrogenase assay and the same amount of PDC activity was used in the series identified above.

Table 3 indicates the substance volumes used in \ mul. Each reaction series was performed by tripled to pH values 6.0, 6.4 and 7.0. The pH value was adjusted by means of the buffer of the substrate solution B3OP. The activity of the PDC was about 2.5 units / ml at pH 7. The substrate and the Enzyme concentrations are also evident from the table 3 below. A sample of 100 µl was taken after 10 minutes, 30 minutes, 60 minutes and 120 minutes, and the reaction is stopped by adding 100 µL of 1M NaOH. The Quantification of B3AP concentration was performed using CE (capillary electrophoresis).

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TABLE 3

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Table 4 below shows the conversion to different pH values for the different PDCs. It is clear that the use of the PDC increases the conversion. It is also evident that the PDC of Z. palmae (Zpa) causes a somewhat higher conversion than the PDC of Z. mobilis (Zmo). It is also evident that lower pH values, such as 6.0 or 6.4, lead to a marked increase in the conversion in the series that use PDC, which is not observed in the control without PDC. In all the reaction series it could be observed that after 120 minutes the conversion decreased.

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TABLE 4

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Example 4 Stability of B3AP in the presence of several reactants of one asymmetric synthesis reaction

To show the stability of B3AP in presence of several reactants incubations of 1 mM B3AP were performed in a reaction tube for 3 hours in the presence of various substances, as indicated in Table 5 below.

The example was performed at a pH of 6 and 7 (buffer sodium phosphate). Immediately after reacting substances  an initial To sample was taken, and another T1 sample after 3 hours. After extraction the concentration of amine was determined with an internal standard (αMBA) by CE. From the Unlike the concentrations obtained, the% of reduction of B3AP concentration (see Figure 3).

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TABLE 5

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Figure 3 shows that the different reagents do not significantly affect the concentration of B3AP (series from 1 to 9). The reaction series from 11 to 13 show the Acetaldehyde influence. Series 11 shows that in absence of a transaminase there is no reduction in the concentration of B3AP, while in the presence of a transaminase and acetaldehyde a strong reduction in the concentration of B3AP can be observed. Acetaldehyde works in the transaminase reaction as an acceptor amino, like pyruvate, and obviously results in a reduction in B3AP concentration.

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Example 5 Reaction of B3AP with the amino pyruvate and acetaldehyde acceptors (does not belong to this illustration)

In this example, the transaminase activity of the flu-TA of V. fluvialis and A. denitrificans in the substrates B3AP and pyruvate, and in B3AP and acetaldehyde is shown.

2 mM B3AP was reacted with 2 mM pyruvate or 2 mM acetaldehyde (36 µl of Alcaligenes denitrificans (Ade) transaminase or 6 µl of Vibrio fluvialis transaminase (Vfl) were reacted per 0.5 µl volume reaction, corresponding to 2 units / µl of transaminase, for 30 minutes). Figure 4 shows the results. Accordingly, B3AP was converted to B3OP by both enzymes, both with pyruvate and acetaldehyde, without any significant difference.

Example 6 Asymmetric synthesis of B3AP under reduced pressure at pH 6

In this example, a reduced pressure was applied to the reaction mixture for an asymmetric synthesis reaction to form B3AP. As a control, the same reaction was performed under normal pressure and without PDC.

Reaction conditions:

Final Volume: 1.5 ml

Phosphate buffer 50 mM sodium, pH 6

300 µl transaminase from Vibrio fluvialis

60 \ mul Zpa-PDC (correspond to 8 units / ml at pH 7)

B3OP 5 mM

D, 10 mM L-alanine

TPP, PLP 0.1 mM

MgCl2 5 mM

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The reduced pressure was applied using a rotary evaporator (150 mBar). The measurement was made using EC.

Figure 5 shows the conversion of B3OP to B3AP over time at different pressures. It is evident that the Conversion is virtually independent of the pressure applied. The conversion at a pressure of 150 mbar is, with respect to the maximum conversion achieved, similar to conversion at a pressure of 1000 mbar

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Example 7 Comparison of several pyruvate decarboxylases

In this example, three different pyruvate decarboxylases were used for the asymmetric synthesis of B3AP. The reaction conditions correspond to those of example 6, except for the use of a pH of 7. The PDCs of Z. mobilis, Z. palmae and a PDC of Biocatalytics (catalog number PDC-101) were used. The activities of the PDCs in the ADH trial were identical (1.6 units / ml).

Figure 6 shows that the three PDCs result in essentially comparable conversions.

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Example 8 Influence of enzyme concentrations and co-substrate in the conversion of B3OP to B3AP

This example shows the influence of the PDC concentration on the conversion of B3OP to B3AP using Alanine as amino donor. In addition, the influence of the Alanine concentration in the conversion of B3OP to B3AP.

The reaction conditions are provided in Example 6, except for the use of a pH of 7 and unless otherwise indicated. The Zymobacter palmae TA was used.

As evidenced from Figure 7, in a reaction without PDC, a 5-fold increase in the concentration of Alanine, from 5 mM to 25 mM, results in a doubling of the conversion (conversion of 12% vs. 5.3% after 2 hours). In presence of PDC, the conversion increases to double with a 5-fold alanine excess (30% vs. 17% after 90 minutes) In the event that the amount of PDC is increased to the usual alanine concentration of 1.6 units / ml at 50 units / ml, the conversion also increases, however only by one <2 factor (29% versus 17% after 40 minutes).

In an additional series of experiments, shows the influence of a combination of excess alanine and excess of PDC, both at a pH of 6 and 7. The reaction is more fast at pH 6, while after reaching a 49% conversion, B3AP concentration also decreases more rapidly. At a pH of 7 the conversion reaches 56%.

Example 9 Influence of alanine concentration on synthesis asymmetric B3AP

In four reactions, they were used alanine concentrations of 5 mM, 25 mM, 110 mM, 300 mM and 500 mM. For each series, a control without PDC and a reaction with PDC The pH value was adjusted to pH 7.0. Samples were taken every half an hour for a reaction time of 3 hours. The times of Conversion reaction provided in Figure 8 were of 40 minutes at 5 mM, 60 minutes at 25 mM and 90 minutes from 110 to 500 mM.

Figure 8 shows the synthesis conversion asymmetric B3AP at high concentrations of alanine.

Figure 8 clearly shows that the conversion It can reach 60 to 70%. It is evident that increasing Alanine concentration of 25 to 110 mM has an effect significant about the conversion of B3OP to B3AP and that a higher increase up to 500 mM only slightly influence the conversion. The PDC's influence on the conversion is reduced as Alanine concentration increases.

From the control reaction data, it was calculated B3AP synthesis equilibrium constant as follows:

7

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Y

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8

Thus, using the measured B3AP concentration, The equilibrium constant was calculated as:

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9

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TABLE 6

10

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Table 6 shows the calculated values. So the equilibrium constant for the reaction with B3AP is 3.1 x 10 3. In this way, the B3OP substrate is a suitable substrate for asymmetric synthesis unlike other ketones.

Example 10 Influence of PLP on the asymmetric synthesis of B3AP

In this example, the influence of PLP is shown (pyridoxal-5'-phosphate) in the B3OP to B3AP conversion. The following series of analyzes were analyzed. reactions.

a) Series 1 using 0.1 mM PLP without adding PLP additional.

b) In series 2, the PLP was added during the reaction as soon as the yellow color begins to disappear, which is due to the presence of PLP in the reaction medium. With this purpose, 1 to 2 µl of a saturated solution of PLP, thus maintaining a strong yellow color. The influence on the concentration of amine through this slight increase in volume is considered to be below 1% and may, so Therefore, consider yourself negligible.

c) No PLP present and no PLP added.

Reaction conditions: 1 ml of final volume, 37 µl of Vfl-transaminase, 5 mM B3OP, buffer pH 7.0 phosphate. The concentration of L-alanine was 110 mM. The measurements taken by CE and α-MBA were used as standard internal.

Figure 9 shows the conversion along the weather. There does not seem to be a significant influence on the maximum conversion due to the addition of PLP in series b) in comparison with series a). The reaction without PLP seems to be more slow, although it reaches the same conversion as the rest of reactions. The addition of PLP in series b) causes a slight greater reduction in amine concentration when compared to The control series.

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Example 11 Asymmetric synthesis of B3AP with acetaldehyde removal by adding nitrogen

The following example details a way of improve the conversion of asymmetric synthesis of B3AP through the acetaldehyde removal.

Reaction conditions:

Substrates:

B3OP 5 mM

500 mM L-Alanine

Zpa-PDC 32 U / ml

Vfl-transaminase 37 \ mul / ml

Phosphate buffer sodium pH 7.0

0.1 mM PLP and TPP (thiamine diphosphate)

MgCl2 5 mM

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Since the reaction solution contains a significant amount of protein, there is a strong tendency to foaming To remove said foam, 0.6 was added µl of an antifoam concentrate A (Sigma, polymer of silicone) to the reaction series. The concentrate suppressed the foam generation largely but not inhibited by full. To exclude that said antifoam concentrate inhibits enzymes, a control series was supplemented without the addition of nitrogen with antifoam A.

Since the addition of dry nitrogen leads to an evaporation of water from the reaction solution, the nitrogen is moistened.

Figure 10 shows the relative conversions calculated from B3AP. The control with antifoam A (N2 -control: Vfl-TA, Zpa-PDC, antifoam, without nitrogen N2) without the nitrogen addition corresponds exactly to the series of the reaction without antifoam A (Vfl-TA, ZpaPDC). So, enzymes are not influenced by the addition of the concentrate of antifoam The series (N2 series: Vfl-TA, Zpa-PDC, antifoam, N2) treated with nitrogen showed an increase in conversion after 60, 90 and 180 minutes.

Example 12 Asymmetric synthesis of B3AP under various conditions

Reaction conditions:

Final Volume: 1 ml

3.7 \ mul of Vfl-transaminase

32 units / ml of Zpa-PDC or PDC Biocatalytics or 3.2 units / ml of Zmo PDC

B3OP 5 mM

500 mM L-Alanine

PLP cofactors 0.1 mM and TPP, 5 mM MgCl2

pH 7, phosphate sodium

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Figure 10 shows the conversion of B3OP to B3AP for the reaction series identified above that combine Vfl-transaminase with each PDC.

In Figure 10 the designated series N2 is the series containing Vfl-transaminase and Zpa-PDC treated with nitrogen and antifoam A. The N2 control is a sample that contains Vfl-transaminase, Zpa-PDC and antifoam A without nitrogen treatment. It is evident that there is a significant increase in the conversion of the N2 control into the N2 sample due to the presence of nitrogen, which was injected into The reaction solution. Thus, by injecting nitrogen in its form gaseous in the reaction medium significantly increases the B3OP to B3AP conversion.

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Example 13 Asymmetric synthesis of B3AP in the presence of alcohol dehydrogenase (ADH)

To eliminate acetaldehyde produced by the PDC reaction of the reaction mixture, ADH can be used to convert acetaldehyde into ethanol.

Reaction conditions:

110 mM L-Alanine

B3Op 5 mM

37 µl / ml of Vfl-transaminase

32 units / ml Zpa PDC PLP 0.1 mM and TPP

MgCl2 5 mM

Phosphate buffer sodium, pH 7

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The following series of reaction:

Reaction series 1: reaction with PDC and transaminase

Reaction series 2: reaction with PDC and 5 mM ethanol transaminase (final concentration) and addition of NADH

Reaction series 3: reaction with PDC, ADH and NADH

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The ADH used was the Saccharomyces cerevisae ADH with an activity between 50 and 100 units / ml. PDC activity was 32 units / ml.

At the start of the reaction, ethanol was added absolute to reaction series 2 in a final concentration of 5 mM. At the start of the reaction, 5 µl of NADH was added to the reaction series 2 and 3 corresponding to a final concentration of 5 mM NADH. After 10 minutes, another 2.4 µmol of NADH was added (4 µL of a 0.6 M solution of NADH in 50 mM phosphate buffer, pH 8.5). The NADH solution was stored on ice and prepared immediately before using it.

The results are provided in Figure 11 and Table 5. The effect of ADH is very evident. The conversion increases to approximately 90%. The control 2 series without ADH and 5 mM ethanol only slightly deviated from the control series 1. Thus, the addition of ADH greatly increases the conversion in the synthesis asymmetric of B3AP from B3OP.

Claims (18)

1. A process for the preparation of an amine optically active chiral and pyruvate as a byproduct alpha-ketone comprising: a) providing a amino acceptor and alanine as amino donor, b) react the amino acceptor and the amino donor with a selective transaminase of (R) or (S), c) obtain the desired optically active chiral amine and pyruvate as an alpha-ketone by-product and d) eliminate the pyruvate of the reaction mixture with a decarboxylase. 2. The process according to claim 1, wherein the decarboxylase is a pyruvate decarboxylase (PDC). 3. The process according to claim 2, in which the acetaldehyde formed by the action of the PDC is eliminates 4. The process according to claim 3, in which acetaldehyde is removed by its reaction with at Less an enzyme. 5. The process according to claim 4, in which the enzyme is an alcohol dehydrogenase. 6. The process according to claim 3, in which acetaldehyde is removed by feeding with nitrogen gaseous reaction mixture. 7. The process according to claim 3, in which acetaldehyde is removed by applying a pressure reduced to the reaction mixture. 8. The process according to claim 3, in which acetaldehyde is removed by methods Chemicals 9. The process according to any of the preceding claims, wherein the amino acceptor is select from the group consisting of phenylpyruvic acid, a salt thereof, pyruvic acid, a salt thereof, acetophenone, 2-ketoglutarate, 3-oxobutyrate, 2-butanone, 3-oxopyrrolidine (3-OP), 3-pyridylmethyl ketone (3-PMK), ethyl acid ester 3-oxobutyric (3-OBEE), ester 3-oxopentanoic acid methyl (3-OPME), N-1-boc-3-oxopiperidinone, N-1-boc-3-oxopyrrolidine (B30P), 3-oxo-piperidine, alkyl-3-oxo-butonoates, Methoxyacetone and 1-Oxotetralone. 10. The process according to any of the preceding claims, wherein the amines obtained are monocyclic or bicyclic amines, in particular cyclic amines of 5 to 6 members or heterocyclic hydrocarbons S-, O- or N-substituted or aromatic amines, in particular aromatic amines substituted with alkyl or alkoxy. 11. The process according to any of the preceding claims, wherein the obtained amines are selected from the group consisting of phenylalanine, alanine,  3-aminopiperidine, alkyl-3-amino-buta-noatos,  3-aminopyrrolidine (3-AP), 3-pyridyl-1-ethylamine (3-PEA), N-1-boc-3-aminopyrrolidine (B3AP), 3-aminobutyric acid ethyl ester (3-ABEE), methyl acid ester 3-aminopentanoic acid (3-APME), α-methylbenzylamine (α-MBA), 1-aminotetralin, α-methyl-4- (3-pyridyl) -butamine, glutamate, β-aminobutyrate, sec-butylamine, methoxyisopropylamine, derivatives of 3-aminopyrrolidine, 1-N-boc-3-aminopiperidine, cephalosporin and cephalosporin derivatives. 12. The process according to any of the preceding claims, wherein the? -Transaminase is from Vibrio fluvialis, Alcaligenes denitrificans, Klebsiella pneumoniae or Bacillus thuringiensis . 13. The process according to any of the preceding claims, wherein the chiral amine optically active obtained in steps c) or d) is removed from the mixture of reaction. 14. The process according to any of the preceding claims, wherein the process is carried out in a reaction mixture with a pH of 5.0 to 9.5. 15. The process according to any of the preceding claims, wherein the process is performed for a reaction time of 40 to 70 minutes. 16. The process according to any of the preceding claims, wherein the reaction is carried out in presence of at least one pyruvate decarboxylase and at least one alcohol dehydrogenase 17. The process according to any of the preceding claims, wherein the reaction is carried out in presence of at least one pyruvate decarboxylase, at least one alcohol dehydrogenase and additionally in the presence of nitrogen gas (N2). 18. A process for the preparation of a physiologically active compound selected from the group of 3-aminopyrrolidone derivatives, cephalosporin, cephalosporin derivatives, heterocyclic boronic acids, L-dihydroxyphenylalanine (LDopa), α-methyldopa, D-phenylglycine, β-hydroxyphenylglycine, phosphinothricin, pyrimido derivatives and pyrrolidone derivatives, in which the process of any one of claims 1 to 17.